TY - JOUR
T1 - Detection of tumor-derived extracellular vesicles in plasma from patients with solid cancer
AU - Vitale, Silvia R.
AU - Helmijr, Jean A.
AU - Gerritsen, Marjolein
AU - MC, Erasmus
AU - van Dessel, Lisanne F.
AU - Beije, Nick
AU - van der Vlugt-Daane, Michelle
AU - Vigneri, Paolo
AU - Sieuwerts, Anieta M.
AU - Dits, Natasja
AU - van Royen, Martin E.
AU - Jenster, Guido
AU - Sleijfer, Stefan
AU - Lolkema, Martijn
AU - Martens, John W.M.
AU - Jansen, Maurice P.H.M.
N1 - Funding Information:
We thank Jeroen van de Peppel (Erasmus MC) for his support with the NTA.
Funding Information:
This work was supported in part by KWF-Alpe d’Huzes project [EMCR2014–6340], an Erasmus MC MRace pilot grant, and in part by a grant from Cancer Genomics Netherlands (CGC.nl)/Netherlands Organization for Scientific Research (NWO).
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/3/24
Y1 - 2021/3/24
N2 - Background: Extracellular vesicles (EVs) are actively secreted by cells into body fluids and contain nucleic acids of the cells they originate from. The goal of this study was to detect circulating tumor-derived EVs (ctEVs) by mutant mRNA transcripts (EV-RNA) in plasma of patients with solid cancers and compare the occurrence of ctEVs with circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA). Methods: For this purpose, blood from 20 patients and 15 healthy blood donors (HBDs) was collected in different preservation tubes (EDTA, BCT, CellSave) and processed into plasma within 24 h from venipuncture. EVs were isolated with the ExoEasy protocol from this plasma and from conditioned medium of 6 cancer cell lines and characterized according to MISEV2018-guidelines. RNA from EVs was isolated with the ExoRNeasy protocol and evaluated for transcript expression levels of 96 genes by RT-qPCR and genotyped by digital PCR. Results: Our workflow applied on cell lines revealed a high concordance between cellular mRNA and EV-RNA in expression levels as well as variant allele frequencies for PIK3CA, KRAS and BRAF. Plasma CD9-positive EV and GAPDH EV-RNA levels were significantly different between the preservation tubes. The workflow detected only ctEVs with mutant transcripts in plasma of patients with high amounts (> 20%) of circulating tumor DNA (ctDNA). Expression profiling showed that the EVs from patients resemble healthy donors more than tumor cell lines supporting that most EVs are derived from healthy tissue. Conclusions: We provide a workflow for ctEV detection by spin column-based generic isolation of EVs and PCR-based measurement of gene expression and mutant transcripts in EV-RNA derived from cancer patients’ blood plasma. This workflow, however, detected tumor-specific mutations in blood less often in EV-RNA than in cfDNA.
AB - Background: Extracellular vesicles (EVs) are actively secreted by cells into body fluids and contain nucleic acids of the cells they originate from. The goal of this study was to detect circulating tumor-derived EVs (ctEVs) by mutant mRNA transcripts (EV-RNA) in plasma of patients with solid cancers and compare the occurrence of ctEVs with circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA). Methods: For this purpose, blood from 20 patients and 15 healthy blood donors (HBDs) was collected in different preservation tubes (EDTA, BCT, CellSave) and processed into plasma within 24 h from venipuncture. EVs were isolated with the ExoEasy protocol from this plasma and from conditioned medium of 6 cancer cell lines and characterized according to MISEV2018-guidelines. RNA from EVs was isolated with the ExoRNeasy protocol and evaluated for transcript expression levels of 96 genes by RT-qPCR and genotyped by digital PCR. Results: Our workflow applied on cell lines revealed a high concordance between cellular mRNA and EV-RNA in expression levels as well as variant allele frequencies for PIK3CA, KRAS and BRAF. Plasma CD9-positive EV and GAPDH EV-RNA levels were significantly different between the preservation tubes. The workflow detected only ctEVs with mutant transcripts in plasma of patients with high amounts (> 20%) of circulating tumor DNA (ctDNA). Expression profiling showed that the EVs from patients resemble healthy donors more than tumor cell lines supporting that most EVs are derived from healthy tissue. Conclusions: We provide a workflow for ctEV detection by spin column-based generic isolation of EVs and PCR-based measurement of gene expression and mutant transcripts in EV-RNA derived from cancer patients’ blood plasma. This workflow, however, detected tumor-specific mutations in blood less often in EV-RNA than in cfDNA.
UR - http://www.scopus.com/inward/record.url?scp=85103350189&partnerID=8YFLogxK
U2 - 10.1186/s12885-021-08007-z
DO - 10.1186/s12885-021-08007-z
M3 - Article
C2 - 33761899
AN - SCOPUS:85103350189
SN - 1471-2407
VL - 21
JO - BMC Cancer
JF - BMC Cancer
IS - 1
M1 - 315
ER -
